Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation

International Journal of Molecular Sciences Article Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation Mária Szatmári-Tóth 1, Tanja Ilmarinen 2, Alexandra Mikhailova 2, Heli Skottman 2, Anu Kauppinen 3, Kai Kaarniranta 4,5 , Endre Kristóf1, Lyubomyr Lytvynchuk 6, Zoltán Veréb7, LászlóFésüs 1and Goran Petrovski 1,7,8,* 1Department of Biochemistry and Molecular Biology, University of Debrecen, Faculty of Medicine, 4032 Debrecen, Hungary; [email protected] (M.S.-T.); [email protected] (E.K.); [email protected] (L.F.) 2Tampere University, Faculty of Medicine and Health Technology, 33014 Tampere, Finland; [email protected] (T.I.); [email protected] (A.M.); [email protected] (H.S.) 3School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, Finland; [email protected] 4Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, 70211 Kuopio, Finland; [email protected] 5Department of Ophthalmology, Kuopio University Hospital, 70029 Kuopio, Finland 6Department of Ophthalmology, Justus-Liebig-University Giessen, Eye Clinic, University Hospital Giessen and Marburg GmbH, Campus Giessen, 35390 Giessen, Germany; Lyubomyr.L[email protected] 7Department of Ophthalmology, Faculty of Medicine, University of Szeged, 6720 Szeged, Hungary; [email protected] 8Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo, Kirkeveien 166, 0450 Oslo, Norway *Correspondence: [email protected]; Tel.: +47-9222-6158 Received: 7 November 2018; Accepted: 13 February 2019; Published: 20 February 2019   Abstract: Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy. Keywords: age-related macular degeneration; anoikis; autophagy; hESC-RPE; inflammation; macrophages; phagocytosis; triamcinolone Int. J. Mol. Sci. 2019,20, 926; doi:10.3390/ijms20040926 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019,20, 926 2 of 20 1. Introduction The retinal pigment epithelium (RPE) is a monolayer of polarized, densely pigmented cells located between the neural retina and the choriocapillaris, thus forming the outer blood-retinal barrier (BRB). The RPEs play an essential role in maintaining the homeostasis of the neural retina, including transport of nutrients and metabolites to the photoreceptors, secretion of growth factors, absorption of incident light, and phagocytosis of photoreceptor outer segments [ 1 ]. The dysfunction and death of RPE cells significantly contribute to the progression of age-related macular degeneration (AMD), which is the leading cause of blindness in the elderly of the developed world [2,3]. One of the most promising future treatment strategies for AMD is the replacement of dysfunctional RPE using cell-based transplantation therapy [ 4 ]. Human pluripotent stem cells have unlimited self-renewal characteristics [ 5 , 6 ] and possess the ability to differentiate into functional RPE cells [ 7 ]. The feasibility of this new approach has been studied extensively [ 8 – 10 ]. Recently, some reports on human embryonic stem cell-derived RPE (hESC-RPE) being generated on a transplantable, biopolymer coated polyimide membrane [ 11 ] as well as a parylene membrane [ 12 ] have been provided. However, the immunogenicity of hESC-RPE cells [ 13 ] has been less confirmed, and not much is known about the death and clearance modalities of such cells. To date, several different types of cell death have been observed in the retina, such as apoptosis, anoikis, or autophagy-associated cell death, which have been implicated in the pathogenesis of AMD [ 14 , 15 ]. The accumulation of drusen (yellow deposits) in the space between Bruch’s membrane and the RPE layer leads to elevation of RPE cells from this membrane inducing anoikic cell death due to detachment from the extracellular matrix (ECM) [ 16 ]. On the other hand, the terms “autophagic” [ 17 ] or “autophagy-associated” cell death [ 18 , 19 ] or type II programmed cell death [ 20 ] have been established based on detection of increased autophagic vacuoles in the dying cells. Autophagy is present at a low or basal level in most tissues, and it is stimulated in response to stress conditions, such as insufficiency of nutrients, hypoxia, or oxidative stress [ 21 ]. The microtubule-associated protein light chain 3 (LC3) is considered to be the major marker of autophagy. The conversion of LC3 (LC3-I to LC3-II) can be followed by immunoblot analysis, the amount of the lipidated LC3-II being clearly associated with the number of autophagosomes [ 22 ]. Recently, enhanced autophagic activity was observed in the aging RPE while accumulated autophagy markers could be detected in the drusen of eyes from AMD patients [23]. The clearance of dying cells is fundamental for proper tissue homeostasis and for balance of the innate immune response [ 24 ]. Insufficient removal of dying RPE cells by non-professional phagocytes can result in debris formation and the development of dry-type of AMD [ 25 ]. Furthermore, disruption of the BRB can lead to a more advanced form of this disease, so-called wet-type of AMD, causing the appearance of professional phagocytes, such as macrophages [ 26 , 27 ]. The exposure of “eat-me” signals on the surface of apoptotic cells, such as phosphatidylserine, can mediate the recognition of such dying cells by phagocytes and promote their engulfment [28,29]. Triamcinolone (TC) is a conventional corticosteroid, which has anti-inflammatory and antiangiogenic activity. The intravitreous (TC) injection is a potent therapeutic treatment for inflammatory ocular diseases including AMD [ 30 – 33 ]. We have previously shown that TC treatment of macrophages results in enhanced clearance of anoikic RPE cells in vitro [ 34 ]. In addition, recent observations have suggested that phagocytic uptake of autophagy-associated dying cells by macrophages triggers a pro-inflammatory response, characterized by the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-8 cytokines from macrophages [35]. It is widely accepted that increased low-level chronic inflammation is strongly associated with AMD; the levels of many inflammatory mediators have been found to be increased in AMD patients. These mediators are responsible for the appearance of inflammatory cells, activation of the inflammasome, promotion of neovascularization and the modulation of inflammatory processes [ 36 ]. In the current study, we aimed to establish an in vitro detection model for studying anoikic and autophagy-associated cell death in hESC-RPEs with implications to AMD. The clearance of Int. J. Mol. Sci. 2019,20, 926 3 of 20 anoikic and autophagy-associated dying RPE cells by professional phagocytes was studied using flow cytometry analysis. Additionally, the released pro-inflammatory cytokines during phagocytosis of dying hESC-RPE cells were investigated with relevance to immune tolerance, serving as a model for studying AMD in vitro and for future stem cell therapy. 2. Results 2.1. Anoikis is Induced in hESC-RPE Cells Anoikis is a form of programmed cell death which is crucial for the maintenance of tissue homeostasis [ 16 ]. The hESC-RPE cells were plated on poly-2-hydroxyethylmethacrylate (poly-HEMA)- coated culture dishes for 24 h to induce anoikic cell death. The typical cobblestone RPE cell morphology [ 37 , 38 ] and high degree of pigmentation could be detected in untreated (control) hESC-RPE cells by phase-contrast microscopy. The representative phase contrast image of anoikic hESC-RPE cells shows the formation of floating aggregates (Figure 1A). The cell death rate could be assessed by Annexin V-fluorescein isothiocyanate (FITC)/propidium-iodide (PI) double staining assay using flow cytometry analysis. Representative dot plots demonstrate the gated regions based on forward light scattering (FSC; X axis), which indicates the cell size, and side light scattering (SSC; Y axis), which indicates the cell granularity (Figure 1B, top dot plots). The viable cells are both annexin-V and PI negative (lower left quadrant) and the annexin-V/PI double positive (upper right quadrant) dots indicate late apoptosis (Figure 1B, bottom dot plots). Significantly lower percentage of viable cells in anoikis-induced hESC-RPE cells compared to the control untreated cells could be detected: it decreased from 83.97 ± 4.64% to 58.5 ± 5.56%. In parallel, the ratio of only annexin V positive, early apoptotic hESC-RPE cells significantly increased from 12.9 ± 3.88% to 36.41 ± 4.15% as a result of ECM detachment. In case of anoikic cells 3.47 ± 0.66% double positive, late apoptotic cells could be detected, while the untreated controls contained only 2.38 ± 1.01% of double positive cells (Figure 1C). 2.2. hESC-RPE Cells Die Due to Serum Deprivation and H2O2Co-Treatment The untreated control hESC-RPE cells formed confluent monolayers with highly pigmented cobblestone morphology in the presence of serum replacement, while these monolayers could be disrupted as a result of serum deprivation (2 h). H 2 O 2 treatment (2 h, 1 mM) in the presence of serum led to swelling and detachment of hESC-RPE cells, while serum deprivation and H 2 O 2 (2 h, 1 mM) co-treatment further increased the rate of cell detachment and dead cell aggregates’ formation (Figure 2A). The cell viability was checked after serum deprivation and H 2 O 2 co-treatment by Annexin V-FITC/PI assay using flow cytometry. Representative dot plots showed the viable (AnxV − /PI − ), early apoptotic (AnxV + /PI − ), necrotic (AnxV − /PI + ) and late apoptotic (AnxV + /PI + ) cell populations upon different conditions (Figure 2B). Serum deprivation and H 2 O 2 co-treatment in hESC-RPE cells resulted in a significantly decreased percentage of viable cells compared to the untreated controls: it decreased from 81.19 ± 1.94% to 47.61 ± 10.63%. Simultaneously, in response to the co-treatment, significantly higher percentage of annexin V single positive hESC-RPE cells (44.91 ± 9.90%) was observed compared to untreated controls (15.68 ±1.60%) (Figure 2C). Int. J. Mol. Sci. 2019,20, 926 4 of 20 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 21 Figure 1. Morphological and cell death analysis after blocking the attachment of human embryonic stem cells-derived retinal pigment epithelium (hESC-RPE) cells to extracellular matrix (ECM). (A) Phase contrast images (10×) of untreated control hESC-RPE cells and anoikic hESC-RPE cells which were cultured on poly-2-hydroxyethylmethacrylate (poly-HEMA) coated culture dishes for 24 h to induce cell death by detachment from the extracellular matrix. Images were captured with a Nikon Eclipse TE2000-S phase contrast microscope. Scale bar indicates 20 µm. (B) The induction of cell death by anoikis was determined by Annexin (Anx)V-FITC/PI double staining assay. Representative dot plots of AnxV/PI measurements of anoikic dying hESC-RPE cells are shown. Top: dot plots Figure 1. Morphological and cell death analysis after blocking the attachment of human embryonic stem cells-derived retinal pigment epithelium (hESC-RPE) cells to extracellular matrix (ECM). ( A ) Phase contrast images (10 × ) of untreated control hESC-RPE cells and anoikic hESC-RPE cells which were cultured on poly-2-hydroxyethylmethacrylate (poly-HEMA) coated culture dishes for 24 h to induce cell death by detachment from the extracellular matrix. Images were captured with a Nikon Eclipse TE2000-S phase contrast microscope. Scale bar indicates 20 µ m. ( B ) The induction of cell death by anoikis was determined by Annexin (Anx)V-FITC/PI double staining assay. Representative dot plots of AnxV/PI measurements of anoikic dying hESC-RPE cells are shown. Top: dot plots represent the measurements of forward light scattering (FSC; X axis) vs. side light scattering (SSC; Y axis). Bottom: the horizontal axis represents the intensity of staining for Annexin V (log scale) and the vertical axis Int. J. Mol. Sci. 2019,20, 926 5 of 20 shows the intensity of staining for PI (log scale). The numbers in the quadrants indicate the percentage of different cell populations. Cells in the lower left quadrant (AnxV − /PI − ) are viable, those in the lower right quadrant (AnxV + /PI − ) are early apoptotic, those in the upper left (AnxV − /PI + ) are necrotic and those in the upper right (AnxV + /PI + ) are late apoptotic cells. Data are representative of 3 independent experiments. ( C ) The bar charts indicate the average percentage of AnxV − /PI − (black bars), AnxV + /PI − (grey bars), AnxV − /PI + (white bars) and AnxV + /PI + (striped bars) cells from 3 independent experiments. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 21 Figure 2. The effect of serum deprivation and H 2 O 2 co-treatment on the morphology and cell viability of hESC-RPE cells. (A) Phase contrast images (10×) of untreated control, serum-deprived (2 h) and H 2 O 2 -treated (2 h, 1 mM) hESC-RPE cells in the presence or absence of serum. Images were captured with a Nikon Eclipse TE2000-S phase contrast microscope. Scale bar indicates 20 µm. (B) The induction of cell death by anoikis and H 2 O 2 -treatment (2 h, 1 mM) in the presence or absence of serum in hESC-RPE cells was determined by Annexin (Anx)V-FITC/PI double staining assay. Representative dot plots of AnxV/PI measurements of anoikic and H 2 O 2 -treated (2 h, 1 mM) dying hESC-RPE cells are shown. Top: dot plots represent the measurements of forward light scattering (FSC; X axis) vs. side light scattering (SSC; Y axis). Bottom: the horizontal axis represents the intensity of staining for Annexin V (log scale) and the vertical axis shows the intensity of staining for Figure 2. The effect of serum deprivation and H 2 O 2 co-treatment on the morphology and cell viability of hESC-RPE cells. ( A ) Phase contrast images (10 × ) of untreated control, serum-deprived (2 h) and H 2 O 2 -treated (2 h, 1 mM) hESC-RPE cells in the presence or absence of serum. Images were captured Int. J. Mol. Sci. 2019,20, 926 6 of 20 with a Nikon Eclipse TE2000-S phase contrast microscope. Scale bar indicates 20 µ m. ( B ) The induction of cell death by anoikis and H 2 O 2 -treatment (2 h, 1 mM) in the presence or absence of serum in hESC-RPE cells was determined by Annexin (Anx)V-FITC/PI double staining assay. Representative dot plots of AnxV/PI measurements of anoikic and H 2 O 2 -treated (2 h, 1 mM) dying hESC-RPE cells are shown. Top: dot plots represent the measurements of forward light scattering (FSC; X axis) vs. side light scattering (SSC; Y axis). Bottom: the horizontal axis represents the intensity of staining for Annexin V (log scale) and the vertical axis shows the intensity of staining for PI (log scale). The numbers in the quadrants indicate the percentage of different cell populations. Cells in the lower left quadrant (AnxV − /PI − ) are viable, those in the lower right quadrant (AnxV + /PI − ) are early apoptotic, those in the upper left (AnxV − /PI + ) are necrotic and those in the upper right (AnxV + /PI + ) are late apoptotic cells. Data are representative of 3 independent experiments. ( C ) The bar charts indicate the average percentage of AnxV − /PI − (black bars), AnxV + /PI − (grey bars), AnxV − /PI + (white bars) and AnxV+/PI+(striped bars) cells from 3 independent experiments. 2.3. Autophagy is Induced in hESC-RPE Cells Treated by Serum Deprivation and H2O2Co-Treatment Autophagy is a highly regulated process, which can maintain homeostasis by protein degradation and turnover of damaged or unnecessary organelles during new cell formation [ 39 – 41 ]. Under different conditions, autophagy can act to promote cell death through an autophagy-associated process which is distinct from apoptosis; it depends on the level of autophagy activation [ 21 , 42 , 43 ]. The most extensively studied stimuli that induce autophagy are oxidative stress [ 44 , 45 ], starvation or serum-deprivation [ 46 , 47 ]. Detection of increased autophagic markers in dying cells, such as microtubule-associated protein 1 light chain 3 (LC3), serve as an indicator of autophagy-associated cell death. During the process of autophagy, the cytosolic form of LC3 (LC3-I) is converted to the lipidated, autophagosome-membrane-bound form (LC3-II) [ 22 ]. Serum deprivation and H 2 O 2 (2 h, 1 mM) co-treatment was used as an autophagy inducer in hESC-RPE cells. The level of LC3 protein was analyzed by Western blot and the LC3-II/LC3-I ratio was quantified by densitometry. An increased LC3-II/LC3-I ratio could be detected upon such co-treatment compared to untreated control. These data suggest that serum deprivation and H 2 O 2 co-treatment results in induction of autophagy (Figure 3). 2.4. Macrophages can Efficiently Engulf Anokic and Autophagy-Associated Dying hESC-RPE Cells in vitro The phagocytosis of anoikic and autophagy-associated dying hESC-RPE cells by macrophages was analyzed by flow cytometry after 4 h and 8 h of co-incubations, respectively. Furthermore, the effect of TC treatment (48 h, 1 µ M) on the phagocytosis capacity of professional phagocytes was also examined (Figure 4A). The anoikic hESC-RPEs were efficiently removed by macrophages, the average phagocytosis being 32.40 ± 3.45% at 4 h of co-incubation. A more efficient phagocytosis rate was found when autophagy-associated dying hESC-RPE cells were engulfed by macrophages over 8 h of co-incubation: 50.72 ± 2.98%. TC treatment moderately, yet significantly enhanced the engulfing capacity of macrophages for anoikic dying cells (35.15 ± 3.85%), which was similar, yet not significantly increased in case of engulfing autophagy-associated dying hESC-RPEs (Figure 4B). 2.5. The Phagocytosis of Anoikic and Autophagy-Associated Dying hESC-RPE Cells by Macrophages Induces Release of Pro-Inflammatory Cytokines The induction of inflammatory responses in macrophages during engulfment of apoptotic and necrotic cells has been well described [ 28 , 48 – 51 ]. However, to date, only a few studies have investigated the inflammatory effect of clearance of anoikic and autophagy-associated dying cells [ 52 ]. Therefore, the release of pro-inflammatory cytokines by macrophages as a result of uptake of anoikic and autophagy-associated dying hESC-RPE cells in vitro was examined. Anoikic and autophagy-associated dying hESC-RPE cells induced by H 2 O 2 (2 h, 1 mM) were co-incubated with macrophages for 4 h and 8 h, respectively, and the cell culture supernatants were collected for cytokine release study. Int. J. Mol. Sci. 2019,20, 926 7 of 20 In parallel, the anti-inflammatory effect of the glucocorticoid TC (48 h, 1 µ M) on the secretion of IL-6 and IL-8 cytokines during phagocytosis of dying cells by macrophages was monitored (Figure 5). No IL-6 secretion by macrophages could be detected when no interaction with the dying cells occurred (control state). The clearance of anoikic hESC-RPE cells by macrophages resulted in a robust and significant increase in IL-6 secretion (836.33 ± 252.27 pg/mL), which decreased upon TC treatment (780.87 ± 279.18 pg/mL) (Figure 5A). Significantly lower levels of IL-6 release were detected during autophagy-associated dying cells’ uptake (324.37 ± 67.43 pg/mL). Similar secretion pattern for IL-8 was found in comparison to the low amount of IL-8 secreted by TC-treated (120.92 ± 1.90 pg/mL) and untreated (84.40 ± 2.48 pg/mL) macrophages (in absence of dying cells). Interestingly, the engulfment of anoikic cells induced a high increase in IL-8 production (1057.33 ± 416.56 pg/mL) by macrophages, the level of which significantly decreased upon TC-treatment (892.11 ± 442.08 pg/mL). Lower secretion of IL-8 was detected during the clearance of autophagy-associated dying cells (318.13 ± 67.99 pg/mL) compared to anoikic ones, yet this release was significant compared to the background secretion by macrophages alone or in the presence of TC (Figure 5B). TC treatment caused no significant differences in the secretion of IL-6 and IL-8 during phagocytosis of autophagy-associated dying cells by macrophages. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 21 PI (log scale). The numbers in the quadrants indicate the percentage of different cell populations. Cells in the lower left quadrant (AnxV−/PI−) are viable, those in the lower right quadrant (AnxV+/PI−) are early apoptotic, those in the upper left (AnxV−/PI+) are necrotic and those in the upper right (AnxV+/PI+) are late apoptotic cells. Data are representative of 3 independent experiments. (C) The bar charts indicate the average percentage of AnxV−/PI− (black bars), AnxV+/PI−(grey bars), AnxV−/PI+ (white bars) and AnxV+/PI+ (striped bars) cells from 3 independent experiments. 2.3. Autophagy is Induced in hESC-RPE Cells Treated by Serum Deprivation and H 2 O 2 Co-Treatment Autophagy is a highly regulated process, which can maintain homeostasis by protein degradation and turnover of damaged or unnecessary organelles during new cell formation [39–41]. Under different conditions, autophagy can act to promote cell death through an autophagy-associated process which is distinct from apoptosis; it depends on the level of autophagy activation [21] [42,43]. The most extensively studied stimuli that induce autophagy are oxidative stress [44,45], starvation or serum-deprivation [46,47]. Detection of increased autophagic markers in dying cells, such as microtubule-associated protein 1 light chain 3 (LC3), serve as an indicator of autophagy-associated cell death. During the process of autophagy, the cytosolic form of LC3 (LC3-I) is converted to the lipidated, autophagosome-membrane-bound form (LC3-II) [22]. Serum deprivation and H 2 O 2 (2 h, 1 mM) co-treatment was used as an autophagy inducer in hESC-RPE cells. The level of LC3 protein was analyzed by Western blot and the LC3-II/LC3-I ratio was quantified by densitometry. An increased LC3-II/LC3-I ratio could be detected upon such co-treatment compared to untreated control. These data suggest that serum deprivation and H 2 O 2 co-treatment results in induction of autophagy (Figure 3). Figure 3. Autophagy induction as a result of serum deprivation and H 2 O 2 co-treatment in hESC-RPE cells. Representative western blot image for the expression of LC3 in hESC-RPE cells treated with 1 mM H 2 O 2 for 2 h in the presence or absence of serum. Integrated optical density was determined by densitometry for quantification of the LC3-II/LC3-I ratio using the ImageJ software. GAPDH was used as a loading control. Data are representative of three independent experiments. 2.4. Macrophages can Efficiently Engulf Anokic and Autophagy-Associated Dying hESC-RPE Cells in vitro Figure 3. Autophagy induction as a result of serum deprivation and H 2 O 2 co-treatment in hESC-RPE cells. Representative western blot image for the expression of LC3 in hESC-RPE cells treated with 1 mM H 2 O 2 for 2 h in the presence or absence of serum. Integrated optical density was determined by densitometry for quantification of the LC3-II/LC3-I ratio using the ImageJ software. GAPDH was used as a loading control. Data are representative of three independent experiments. Int. J. Mol. Sci. 2019,20, 926 8 of 20 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 21 The phagocytosis of anoikic and autophagy-associated dying hESC-RPE cells by macrophages was analyzed by flow cytometry after 4 h and 8 h of co-incubations, respectively. Furthermore, the effect of TC treatment (48 h, 1 µM) on the phagocytosis capacity of professional phagocytes was also examined (Figure 4A). The anoikic hESC-RPEs were efficiently removed by macrophages, the average phagocytosis being 32.40 ± 3.45% at 4 h of co-incubation. A more efficient phagocytosis rate was found when autophagy-associated dying hESC-RPE cells were engulfed by macrophages over 8 h of co-incubation: 50.72 ± 2.98%. TC treatment moderately, yet significantly enhanced the engulfing capacity of macrophages for anoikic dying cells (35.15 ± 3.85%), which was similar, yet not significantly increased in case of engulfing autophagy-associated dying hESC-RPEs (Figure 4B). Figure 4. The clearance of anoikic and autophagy-associated dying hESC-RPE cells by macrophages. ( A ) Representative flow cytometry dot plots demonstrating phagocytosis of anoikic and autophagyassociated dying hESC-RPE cells by macrophages after 4 h and 8 h co-incubation, respectively. Macrophages were pre-treated with 1 µ M triamcinolone (TC) for 48 h. The horizontal axis represents the intensity of staining for CFDA (log scale) and the vertical axis shows the intensity of staining for CMTMR (log scale). Cells in the upper right quadrant indicate the engulfed hESC-RPE (CFDA-labeled) cells by macrophages (CMTMR-labeled). Data are representative of 3 independent experiments. ( B ) The phagocytosis rate of anoikic and autophagy-associated dying hESC-RPE cells by untreated and TC-pre-treated (48 h, 1 µ M) macrophages after 4 h and 8 h co-incubation, respectively, is shown as determined by flow cytometry analysis. Bars represent the mean ± SD of 3 independent experiments, *p< 0.05. Int. J. Mol. Sci. 2019,20, 926 9 of 20 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 21 Figure 5. Determination of IL-6 and IL-8 release during the engulfment of anoikic and autophagy-associated dying hESC-RPE cells by macrophages. Anoikic dying hESC-RPE cells (left panels) and autophagy-associated dying hESC-RPE cells (right panels) were co-incubated with untreated and triamcinolone (TC)-treated (48 h, 1 µM) macrophages for 4 h and 8 h, respectively, then the supernatants were collected, and the level of secreted IL-6 (A) and IL-8 (B) cytokines were measured by ELISA. Bars represent the mean ± SD of 3 independent experiments, * p < 0.05. 3. Discussion Figure 5. Determination of IL-6 and IL-8 release during the engulfment of anoikic and autophagyassociated dying hESC-RPE cells by macrophages. 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